Project Summary: The objective of this grant is to investigate new mechanisms by which EGFR signaling is regulated. This research will be carried out in Drosophila, which provides an ideal model system for the proposed research because of the well-characterized developmental programs, the large number of reagents available, and the ease of combining genetic, molecular, cellular, and developmental approaches to carry out in vivo studies at single cell resolution. Ligands for the EGFR signaling are often synthesized as membrane tethered precursors and require proper intracellular trafficking and processing to be released at proper location to activate EGFR signaling in vivo. Processing of EGFR ligand is mediated by the membrane protease rhomboid (rho), which is the rate limiting component for EGFR signaling activation. During Drosophila eye development, rho is initially expressed in the developing R8 precursors, which is the initial source of EGFR ligands for stepwise photoreceptor recruitment. How rho expression is coordinated with photoreceptor differentiation during eye development is not known. In genetic screens for mutations that modulate the apoptosis of rbf mutant cells, we identified several mutations that regulate EGFR signaling. Preliminary characterization of these mutants reveal novel mechanisms by which EGFR signaling are regulated by endosomal trafficking and by protein acetylation. In addition, our preliminary results suggest that rho expression is repressed by the Rpd3-mediated epigenetic mechanisms in the undifferentiated progenitor cells and that a distinct mechanism is involved in coordinating R8 specification with the restriction of rho expression to the developing R8 precursors during photoreceptor differentiation. In this grant, we will take advantage of the assays we developed to investigate the regulation of EGFR signaling by endosomal trafficking and by protein acetylation. In addition we will characterize the mechanisms that regulate rho expression in the differentiating photoreceptor cells and identify additional factors that mediate the epigenetic repression of rho in the undifferentiated retinal progenitor cells. These studies will reveal novel control of the EGFR signaling and provide novel insights into the mechanism that coordinate the initial photoreceptor neuron specification with the restriction of EGFR ligand to the specified neuron. Since the mechanisms that regulate EGFR signaling are conserved, results obtained from this study will provide novel insights into the control of EGFR signaling in diverse systems and may lead to the development of novel approaches to block deregulated EGFR activation.